Four Free and Easy-To-Use Online Primer Design Tools

Designing and running PCR reactions in the lab has become so commonplace that the number of primer design tools available can be a bit overwhelming for a beginner (or even an experienced molecular biologist!). Below are four of my favorite online programs available to make primer design quick, easy, and effective.

A quick note before we get started: If you’re new to PCR, Bitesize Bio has a plethora of articles for you! Learn how polymerase chain reaction works, what to do when it doesn’t work, and variations on standard PCR. And after that, check out the e-book “The Bitesize Bio Guide to PCR” to become a master of this invaluable molecular biology technique.

1. IDT OligoAnalyzer: This is a simple yet effective tool for determining physical properties of oligo sequences up to 255 bases. After plugging in a sequence, you will get information on sequence length, GC content, melting temperature range, molecular weight, extinction coefficient, and optical density. I find the hairpin structure formation tab particularly fun, and the latest version also allows users to perform an NCBI Blast easily without copying and pasting the sequence.

A word to the wise: check on the primer without any added restriction sites for cloning before ordering your primer. Since the added site doesn’t bind to template DNA in the first round of amplification, you should ensure that the primer properties do not change significantly and inhibit the reaction!

• Good for users who: Are new to primer design, know what target range for GC content and melting temperatures they want, and are amplifying DNA from a plasmid for typical cloning experiments.
• Other considerations: Not high throughput (sequences are analyzed one at a time manually at the time of writing).

2. NCBI’s PrimerBlast (Primer3): Possibly one of the most highly regarded and versatile online tools for primer design and analysis. PrimerBlast was developed by NCBI and combines the tried and true Primer3 platform with Blast capabilities. The latter component is critical if you are amplifying from an organism’s genome and want to minimize nonspecific binding to genes similar to your target sequence within the genome.

To begin, you can either copy/paste a template into PrimerBlast or use an accession number within NCBI’s database. Then, decide which of the many parameters you’d like to specify- like acceptable product lengths, melting temperature range, and exon/intron selection if you are designing primers for mRNA amplification (exons are divided by non-coding introns in the DNA coding for mRNA).

After the sequence is submitted and analyzed you can also sort results by primer length, melting temperature, and more. My favorite part of using PrimerBlast is that it provides a visual interface including where the primers will bind on the template.

• Good for users who: Are amplifying DNA from a genome, or have a primer already designed and want to analyze its specificity to a template.
• Other considerations: Like IDT’s OligoAnalyzer, this is not a high throughput primer design tool. There is a template limit (50,000 nucleotides) and PrimerBlast doesn’t provide a suggested annealing temperature.

3. BatchPrimer3: This is a great tool for performing a more high-throughput primer design. BatchPrimer3 is based on Primer3 and can design a wide variety of primers. Results are provided as an HTML with color-coded alignment visualization, and can be downloaded in tab-delimited form. With this tool you can also obtain batch information on the suggested primers’ physical parameters.

• Good for users who: Require a high-throughput primer design tool for many template sequences.
• Other considerations: Does not provide suggested annealing temperature and does not automatically BLAST primer results like PrimerBlast does. Also, does not perform an ‘internal sequence test’, meaning that primers are not tested for in silico annealing to templates.

4. HYDEN (HighlY DEgeNerate primers): Sometimes, you don’t know the exact sequence of target DNA you are trying to amplify, like when you are working with an organism’s DNA that has yet to be sequenced. In this case, you may want to design “degenerate” primers based on related organisms or reverse engineering them by using the amino acid sequence. HYDEN is a command-line based downloadable program that designs primers cover many input sequences.

• Good for users who: Don’t know the sequence of template DNA or are trying to amplify from a cDNA library.
• Other considerations: Does not provide physical characteristics of output oligo sequences, not the most user-friendly tool for designing typical cloning primers for known sequences.

Of course, this list isn’t comprehensive – there are many other online options available! A quick word to the wise before plugging in all your favorite templates: do ensure you have a detailed understanding of how PCR works and your specific application prior to designing and ordering primers. While optimal primer design can go a long way at success in the thermocycler, consider varying other key components of your PCR reaction (like annealing temperature, magnesium concentration, and polymerase used) to further improve your odds of success with particularly difficult templates.

What tools do you use for primer design and what do you like about them? Tell us in the comments below!